Binding of NIR-conPK and NIR-6T to astrocytomas and microglial cells: evidence for a proteinrelated to TSPO.

Abstract

PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [(11)C]-PK 11195 and [(11)C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [(3)H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [(3)H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-gamma (but not TNFalpha and TGFbeta) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.

Ability of NIR-conPK and NIR-6T to compete for [3H]-PK 11195 binding to DBT cell homogenates.

(A) Representative specific binding of [3H]-PK 11195 to DBT cell homogenates (average Bmax and Kd are in Table 2). (B) Competition of specific [3H]-PK 11195 binding to DBT cell homogenates by increasing concentrations of PK 11195 (dotted line) or NIR-conPK (solid line). Ki and EC50 values for PK 11195 competition were 41 and 93 nM respectively, and calculated using three independent experiments, each performed in triplicate. Ki value for NIR-conPK could not be reliably calculated, indicating that this MI agent does not bind to TSPO. (C) Competition of specific [3H]-PK 11195 binding to DBT cell homogenates by increasing concentrations of 6T (dotted line) and NIR-6T (solid line). Ki and EC50 values for 6T were 3.6 and 114 nM respectively and for NIR-6T 400 and 412 nM and were calculated using three independent experiments, each performed in triplicate.

(A, B) Kinetics of the total binding when labeling intact DBT cells with 100 nM of either NIR-conPK (A) or NIR-6T (B). (C, D) Ability of PK 11195 and 6T to compete for the binding of (C) NIR-conPK and (D) NIR-6T in intact DBT cells. DBT cells grown in 96 well plate were pre-incubated for 15 min with either vehicle (media with DMSO, i.e. total), PK 11195 (PK, 10 µM) or 6T (DAA, 10 µM), and then incubated with either (A) NIR-conPK (100 nM) or (B) NIR-6T (100 nM) for one hour. Cells were then rinsed and RFU at 800 nm read with an LI-COR Odyssey® Infrared Imaging system. Values are mean±SEM of nine measurements (i.e. three independent experiments, each performed in triplicate). *p<0.01, significantly different from total, as determined by ANOVA followed by Dunnett's. (E, F) Kd of NIR-conPK when using either (E) PK 11195 (10 µM) or (F) DAA1106 (10 µM) to determine non-specific binding. DBT cells grown in a 96 well plate were pre-incubated for 15 min with either media alone (i.e. total), or addition of PK 11195 or DAA1106, and then incubated with increasing concentration of NIR-conPK for one hour. Cells were then rinsed and RFU at 800 nm read with an LI-COR Odyssey® Infrared Imaging system. Values are mean±SEM of nine measurements (i.e. three independent experiments performed in triplicate). Solid line represents specific binding; dotted line with square shows total binding and dotted line with triangle shows non-specific binding.

Primary mouse microglia were incubated for 18–24 hrs with either vehicle (basal), TNFα (5 ng/ml), IFNγ (100 Ui/ml), TNFα plus IFNγ, or TGFβ2 (1 ng/ml). Supernatant aliquots were recovered and amounts of (A) IL-1α, (B) IL-6 and (C) RANTES determined by Luminex beads. Values are mean±SEM of six measurements (i.e. three independent experiments, each performed in duplicate). **p<0.01; *p<0.05, significantly different from basal, as determined by ANOVA followed by Dunnett's.